U.S. patent application number 17/497894 was filed with the patent office on 2022-01-27 for lactic acid bacteria, and feed, fertilizers, and live bacterial preparations containing the same.
The applicant listed for this patent is Takashi OHKUBO. Invention is credited to Takashi OHKUBO.
Application Number | 20220024832 17/497894 |
Document ID | / |
Family ID | |
Filed Date | 2022-01-27 |
United States Patent
Application |
20220024832 |
Kind Code |
A1 |
OHKUBO; Takashi |
January 27, 2022 |
LACTIC ACID BACTERIA, AND FEED, FERTILIZERS, AND LIVE BACTERIAL
PREPARATIONS CONTAINING THE SAME
Abstract
An object is to provide lactic acid bacteria that can be
applicable to feed, fertilizers, live bacterial preparations,
probiotics, etc. The lactic acid bacteria are identified and
deposited under the accession numbers of NITE (National Institute
of Technology and Evaluation) P-02313, NITE P-02314, NITE P-02490,
NITE P-02491, and NITE P-02492. In addition, another object is to
provide feed, fertilizers and live bacterial preparations
containing the lactic acid bacterium/bacteria of the invention.
According to studies, it is confirmed that the lactic acid bacteria
are proliferated in animals including human, plants, etc. without
being destroyed. According to studies, it is also confirmed that
the lactic acid bacteria are proliferated in water, soil, etc.
without being destroyed. Therefore, the lactic acid bacteria can be
widely applicable in feed, fertilizers, live bacterial
preparations, etc.
Inventors: |
OHKUBO; Takashi; (Saga,
JP) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
OHKUBO; Takashi |
Saga |
|
JP |
|
|
Appl. No.: |
17/497894 |
Filed: |
October 9, 2021 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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16332977 |
Mar 13, 2019 |
11174204 |
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PCT/JP2017/032818 |
Sep 12, 2017 |
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17497894 |
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International
Class: |
C05F 11/08 20060101
C05F011/08; A23K 10/18 20060101 A23K010/18; A61K 35/741 20060101
A61K035/741; C12N 1/20 20060101 C12N001/20; A01G 7/06 20060101
A01G007/06 |
Foreign Application Data
Date |
Code |
Application Number |
Sep 13, 2016 |
JP |
2016-178146 |
Jun 30, 2017 |
JP |
2017-128717 |
Claims
1. A lactic acid bacterium deposited under an accession number of
NITE (National Institute of Technology and Evaluation) P-02491,
wherein said lactic acid bacterium is cultured with MRS agar at
30.degree. C. and isolated with a dilution plate method.
2. Feed containing the lactic acid bacterium according to claim
1.
3. A fertilizer containing the lactic acid bacterium according to
claim 1.
4. A live bacterial preparation or probiotic containing the lactic
acid bacterium according to claim 1.
Description
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] This is a divisional application of a prior application Ser.
No. 16/332,977, filed on Mar. 13, 2019, allowed.
BACKGROUND OF THE INVENTION AND RELATED ART STATEMENT
[0002] The present invention relates to lactic acid bacteria. The
present invention also relates to feed, fertilizers and live
bacterial preparations (or probiotics) containing the lactic acid
bacterium.
[0003] Lactic acid bacteria are generally referred to bacteria that
produce lactic acid through metabolic activities thereof. It has
been known that each of lactic acid bacteria tends to exhibit a
different characteristic.
[0004] Conventionally, bacteria with specific characteristics such
as lactic acid bacteria have been extracted so that their
characteristics have been studied to develop products including
drugs, food and drink, etc. taking advantage of such
characteristics. (see Patent Reference for example).
[0005] Patent Reference: Japanese Patent Publication No.
5,597,237
[0006] Generally speaking, conventionally known lactic acid
bacteria are restricted from proliferation by an environmental
condition. Therefore, in many cases, lactic acid bacteria are found
to be not suitable and difficult for making feed, fertilizers, live
bacterial preparations, etc.
[0007] In view of the problem described above, an object of the
present invention is to provide lactic acid bacteria through
diligently engaged researches and studies in extractions of lactic
acid bacteria.
SUMMARY OF THE PRESENT INVENTION
[0008] According to a first aspect of the present invention, a
lactic acid bacterium is identified and deposited under the
accession number of NITE (National Institute of Technology and
Evaluation) P-02313.
[0009] According to a second aspect of the present invention, a
lactic acid bacterium is identified and deposited under the
accession number of NITE P-02314.
[0010] According to a third aspect of the present invention, a
lactic acid bacterium is identified and deposited under the
accession number of NITE P-02490.
[0011] According to a fourth aspect of the present invention, a
lactic acid bacterium is identified and deposited under the
accession number of NITE P-02491.
[0012] According to a fifth aspect of the present invention, a
lactic acid bacterium is identified and deposited under the
accession number of NITE P-02492.
[0013] According to a sixth aspect of the present invention, feed
contains the lactic acid bacterium/bacteria according to any one of
the first to fifth aspect of the present invention.
[0014] According to a seventh aspect of the present invention, a
fertilizer contains the lactic acid bacterium/bacteria according to
any one of the first to fifth aspect of the present invention.
[0015] According to an eighth aspect of the present invention, a
live bacterial preparation (or probiotics) contains the lactic acid
bacterium/bacteria according to according to any one of the first
to fifth aspect of the present invention.
[0016] The lactic acid bacteria of the present invention are
effectively applicable for feed, fertilizers, and live bacterial
preparations.
DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS
Isolation Example 1
[0017] After processing a fish with a special method for
fermentation, the presence of a variety of bacteria was detected in
the fish meat, etc. Among the bacteria thus detected, the presence
of lactic acid bacteria was detected.
[0018] Afterward, it was decided to isolate only the lactic acid
bacteria and conduct the isolation at Techno Suruga Laboratory Co.,
Ltd. (330 Nagasaki, Shimizu-ku, Shizuoka-shi, Shizuoka, Japan).
[0019] To isolate the bacteria, isolation and culturing were
conducted under the following conditions. Thereafter, observation
of the colonies was performed, and gram staining and catalase test
were performed.
Culturing conditions
[0020] Culturing medium: MRS agar (Oxoid, Hampshire, UK) +3%
NaCl
[0021] Culturing temperature: 30.degree. C.
[0022] Culturing period: 3 days
[0023] Diluent: Saline solution
[0024] Dilution factor: Undiluted to 10.sup.4
[0025] Isolation method: Dilution plate method
[0026] Other conditions Anaerobic
Colony observation
[0027] Observed the resultant colonies using a stereo microscope
SZG10 (Olympus, Tokyo, Japan).
Gram staining
[0028] Fiber G "Nissui" (Nissui Pharmaceutical Co., Ltd., Tokyo,
Japan) was used in the gram staining and an optical microscope BX51
(Olympus, Tokyo, Japan) was used as a microscope.
Catalase test
[0029] Using 3% hydrogen peroxide solution, the test was conducted
and it was determined that the result was positive if air bubbles
were produced.
[0030] By the isolation and culturing, growth of a plurality of
colonies having different characteristics was observed. For the
isolated strains, gram staining and the catalase test were
conducted. The bacteria that were gram positive and catalase
negative were determined as lactic acid bacteria. As a result,
colonies of two types of bacteria that were possibly lactic acid
bacteria were isolated. Hereinafter, those bacteria are referred to
as SIID17126-L1 and SIID17126-L2, respectively.
[0031] Next, an identification test was conducted for the
respective bacteria (SIID17126-L1 and SIID17126-L2). The
identification test was conducted by Techno Suruga Laboratory Co.,
Ltd. conducted based on our request for their Bacterial Premium
Test.
[0032] In the identification test, the bacteria were cultured under
the following conditions and 16S rDNA sequence analysis,
morphological observation and physiological/biochemical tests
(bacterial first-stage test and bacterial second-stage test) were
conducted to estimate the group that the bacteria belong to.
Culturing conditions
[0033] Culturing medium: MRS agar (Oxoid, Hampshire, UK)
[0034] Culturing temperature: 30.degree. C.
[0035] Culturing period: 48 hours
[0036] Diluent: Saline solution
[0037] Other conditions Anaerobic
16S rDNA (16S rRNA gene) sequence analysis
[0038] The procedures from PCR amplification to cycle sequencing
were performed based on the respective protocols.
[0039] DNA extraction: [0040] Achromopeptidase (Wako Pure Chemical
Industries, Ltd., Osaka, Japan)
[0041] PCR amplification: [0042] PrimeSTAR HS DNA Polymerase
(Takara Bio Inc., Shiga, Japan)
[0043] Cycle sequencing: [0044] BigDye Terminator v3.1 Cycle
Sequencing Kit (Applied Biosystems, California, U.S.A)
[0045] Primer used this time: [0046] PCR amplification: 9F, 1510R
[0047] Sequencing: 9F, 785F, 802R, 1510R
[0048] Sequencer: [0049] ABI PRISM 3130.times.1 Genetic Analyzer
System (Applied Biosystems, California, U.S.A)
[0050] Sequencing software: [0051] ChromasPro 1.7 (Technelysium Pty
Ltd., Tewantin, AUS)
[0052] BLAST homology search and simplified molecular
phylogenetics: [0053] DNA database for microbial identification D
BOBA10.0 (Techno Suruga Laboratory Co., Ltd., Shizuoka, Japan)
International Nucleotide Sequence Database Collaboration
(GenBank/DDBJ/EMBL) Bacterial first-stage test
[0054] Based on the morphological observation using an optical
microscope BX50F4 (Olympus, Tokyo, Japan) and the method by Barrow
et al. (BARROW, (G.I.) and FELTHAM, (R.K.A.): Cowan and Steel's
Manual for the Identification of Medical Bacteria. 3rd Ed. 1993,
Cambridge University Press), catalase reaction, oxidase reaction,
acid/gas production from glucose, and oxidation/fermentation of
glucose were tested.
Bacterial second-stage test
[0055] For this test, an API 20 Strep kit (bioMerieux, Lyon,
France) was used. Additional experiments were also conducted,
according to technical cooperation matters with NCIMB Ltd. (United
Kingdom, http://www.ncimb.co.uk/) and related references regarding
classification and identification.
[0056] The result of the 16S rDNA sequence analysis suggested that
SIID17126-L1 may belong to Enterococcus, more specifically
E.devriesei, E.pseudoavium, or E.viikkiensis.
[0057] In addition, according to the result of the bacterial
first-stage test, SIID17126-L1 had properties that were consistent
with those of Enterococcus. According to the result of the
bacterial second-stage test, SIID17126-L1 was recognized to have
different properties from those of E. pseudo avium, but have
properties that were consistent with those of E. devriesei or those
of E. viikkiensis.
[0058] Based on the above results, it was estimated that
SIID17126-L1 belongs to Enterococcus viikkiensis or Enterococcus
devriesei.
[0059] In addition, the results of the sequencing analysis of
16SrDNA suggested that SIID17126-L2 may belong to L. latis,
possibly belonging to Lactococcus, more specifically L. lactis
subsp. lactis.
[0060] In addition, according to the results of the bacterial
first-stage test, SIID17126-L1 had properties that were consistent
with those of Lactococcus. According to the results of the
bacterial second-stage test, SIID17126-L1 had properties that were
consistent with those of Lactococcus lactis subsp. lactis.
[0061] Based on the above results, it is determined that
SIID17126-L2 belongs to Enterococcus lactis subsp. lactis.
[0062] Those lactic acid bacteria (SIID17126-L1 and SIID17126-L2)
have been deposited as follows:
[0063] Depository Institution: NITE (National Institute of
Technology and Evaluation) Patent Microorganisms Depository (NPMD)
(2-5-8 Kazusa Kamatari, Kisaradu-shi, Chiba, Japan 292-0818)
[0064] Accession Date: Aug. 2, 2016
[0065] Accession Numbers: NITE P-02313 for SIID 17126-L1 [0066]
NITE P-02314 for SIID 17126-L2
[0067] Here, those lactic acid bacteria have been transferred to
international depository as follows:
[0068] Accession Numbers: NITE BP-02313 for SIID 17126-L1 [0069]
NITE BP-02314 for SIID 17126-L2
[0070] The above-described lactic acid bacteria (SIID17126-L1 and
SIID17126-L2) were confirmed to have the following properties.
Here, detections of the lactic acid bacteria described below were
performed at Japan Food Research Laboratories (52-1 Motoyoyogi-cho,
Shibuya-ku, Tokyo) or at Saga Environmental Science Inspection
Association (1-4-2 Hikari, Saga-shi, Saga).
Isolation Example 2
[0071] Processing fish by a special method for fermentation, the
presence of variety of bacteria was detected in the fish meat, etc.
In the bacteria, the presence of lactic acid bacteria was
detected.
[0072] Accordingly, it was decided to isolate only the lactic acid
bacteria. The isolation of the lactic acid bacteria was performed
by Techno Suruga Laboratory Co., Ltd. (330 Nagasaki, Shimizu-ku,
Shizuoka-shi, Shizuoka, Japan) based on our request.
[0073] In the isolation of the bacteria, isolation and culturing of
the bacteria were conducted under the similar conditions to those
in the above-described Isolation Example 1. Thereafter, observation
of the resultant colonies was performed, and gram staining and
catalase test were conducted.
[0074] By the isolation and the culturing, growth of a plurality of
colonies having different properties was observed. For the isolated
strains, gram staining and the catalase test were conducted. The
bacteria that were gram positive and catalase negative were
determined as lactic acid bacteria. As a result, colonies of three
types of bacteria that were possibly lactic acid bacteria were
isolated. Hereinafter, those bacteria are referred to as
SIID21336-L1, SIID21336-L2 and SIID21336-L3, respectively.
[0075] Next, an identification test was conducted for the
respective bacteria (11D21336-L1, SIID21336-L2 and SIID21336-L3).
The identification test was conducted by Techno Suruga Laboratory
Co., Ltd. based on our request for their Bacterial Premium
Test.
[0076] In the identification test, the bacteria were cultured under
the similar conditions to those of the above-described Isolation
Example 1. Then, 16S rDNA sequence analysis, morphological
observation and physiological/biochemical tests (bacterial
first-stage test and bacterial second-stage test) were conducted to
estimate the genus and species that the bacteria belong to from the
results.
[0077] A result of the 16S rDNA sequence analysis suggested that
SIID21336-L1 may belong to Lactobacillus, more specifically L.
brevis.
[0078] In addition, according to the results of the bacterial
first-stage test, SIID21336-L1 had properties that were consistent
with those of Lactobacillus. According to the results of the
bacterial second-stage test, SIID21336-L1 had consistent properties
to those of L. brevis.
[0079] Based on the above results, it is determined that
SIID21336-L2 belongs to Lactobacillus brevis.
[0080] A result of the 16S rDNA sequence analysis suggested that
SIID21336-L2 may possibly belong to Lactobacillus, more
specifically Lactobacillus sp. that was closely related to L.
plantarum.
[0081] In addition, according to the results of the bacterial
first-stage test, SIID17126-L1 had properties that were consistent
with those of Lactococcus. According to the results of the
bacterial second-stage test, SIID17126-L1 had properties that were
consistent with those of Lactococcus lactis subsp. lactis.
[0082] Based on the above results, it is determined that
SIID21336-L2 belongs to Lactobacillus sp. that is closely related
to L. plantarum.
[0083] A result of the 16S rDNA sequence analysis suggested that
SIID21336-L3 may belong to Enterococcus, more specifically E.
casseliflavus.
[0084] In addition, according to the results of the bacterial
first-stage test, SIID21336-L3 had properties that were consistent
with those of Enterococcus. According to the results of the
bacterial second-stage test, SIID21336-L3 had properties that were
consistent with those of E. casseliflavus.
[0085] Based on the above results, it is determined that
SIID21336-L3 belongs to Enterococcus casseliflavus.
[0086] Those lactic acid bacteria (SIID21336-L1, SIID21336-L2 and
SIID21336-L3) have been deposited as follows:
[0087] Depository Institution: [0088] NITE (National Institute of
Technology and Evaluation) Patent Microorganisms Depositary (NPMD)
(2-5-8 Kazusa Kamatari, Kisaradu-shi, Chiba, Japan 292-0818)
[0089] Accession Date: Jun. 12, 2017
[0090] Accession Numbers: [0091] NITE P-02490 for SIID 21336-L1
[0092] NITE P-02491 for SIID 21336-L2 [0093] NITE P-02492 for SIID
21336-L2
Properties
[0094] The above-described lactic acid bacteria (SIID17126-L1,
SIID17126-L2, SIID21336-L1, SIID21336-L2 and SIID21336-L3) were
confirmed to have the following properties.
[0095] Here, detections of the lactic acid bacteria described below
were performed at Japan Food Research Laboratories (52-1
Motoyoyogi-cho, Shibuya-ku, Tokyo) or at Saga Environmental Science
Inspection Association (1-4-2 Hikari, Saga-shi, Saga).
(1) Feed
[0096] Giving feed not containing lactic acid bacteria to chickens,
and their eggs were tested. As a result, the lactic acid bacteria
were not detected from the eggs (yolks and egg whites).
[0097] In addition, adding representative commercially-available
lactic acid bacteria (yogurt) to the above-described feed that did
not contain the lactic acid bacteria, the feed was given to
chickens and their eggs were tested. As a result, the lactic acid
bacteria were not detected from the eggs (yolks and egg
whites).
[0098] Furthermore, adding the lactic acid bacteria of the
invention (SIID17126-L1, SIID17126-L2, SIID21336-L1, SIID21336-L2
or SIID21336-L3 alone or a mixture of at least two types of them)
to the feed that did not contain lactic acid bacteria, the feed was
given to chicken and their eggs were tested. As a result, the
lactic acid bacterium/bacteria were detected from the eggs (yolks
and egg whites).
[0099] From those results, it was found that the lactic acid
bacteria of the invention (11D17126-L1, SIID17126-L2, SIID21336-L1,
SIID21336-L2 and SIID21336-L3) can proliferate in mother chickens
without being digested or destroyed if added in their bodies, and
can be included in their yolks and egg whites of their eggs.
[0100] As described above, when the above-described lactic acid
bacteria (SIID17126-L1, SIID17126-L2, SIID21336-L1, SIID21336-L2
and/or SIID21336-L3) are contained in feed, the bacteria can
proliferate in animal bodies such as livestock and can be
administered to the animals through eating the feed. In addition,
even their baby birds or eggs, to which the feed was not directly
given, can contain the lactic acid bacteria.
[0101] Accordingly, it can be understood that the feed may be
applicable as a method of producing eggs containing lactic acid
bacteria.
(2) Fertilizer(s)
[0102] Feeding fertilizer not containing the lactic acid bacteria
to tomato seedlings, the resultant ripened tomatoes were tested.
The tomatoes did not contain the lactic acid bacteria.
[0103] However, when the above-described lactic acid bacteria
(SIID17126-L1, SIID17126-L2, SIID21336-L1, SIID21336-L2 or
SIID21336-L3 alone or a mixture of at least two types of them) were
added to the fertilizer that did not contain lactic acid bacteria
and was fed as a fertilizer to tomato seedlings, the lactic acid
bacteria were detected from the resultant ripened tomatoes.
[0104] Moreover, it was also confirmed that the tomatoes ripe
satisfactorily faster than those seedlings, to which the fertilizer
that did not contain the lactic acid bacteria was fed.
[0105] Accordingly, when the above-described lactic acid bacteria
(SIID17126-L1, SIID17126-L2, SIID21336-L1, SIID21336-L2 and/or
SIID21336-L3) are contained in a fertilizer, the lactic acid
bacteria can proliferate inside the plants such as vegetables and
can be administered to the grown plants through the fertilizer. In
addition, the lactic acid bacteria can be also contained in even
their resultant fruits.
[0106] Accordingly, it can be understood that the fertilizer may be
also applicable as a method of producing vegetables containing
lactic acid bacteria.
(3) Live Bacterial Preparation(s) or probiotics
[0107] When the content of lactic acid bacterium was determined in
seawater, river water (fresh water), brackish water (mixture of
seawater and river water), light petroleum-added seawater and
marine soil, lactic acid bacteria were not detected from any of
them.
[0108] To the seawater, river water, brackish water, light
petroleum-added seawater. marine soil, representative
commercially-available lactic acid bacteria (yogurt) was added and
left to stand. As a result, it was confirmed that the added lactic
acid bacteria were destroyed.
[0109] However, when the above-described lactic acid bacteria
(SIID17126-L1, SIID17126-L2, SIID21336-L1, SIID21336-L2 or
SIID21336-L3 alone or a mixture of at least two types of them) were
added to the seawater, river water, brackish water, light
petroleum-added seawater and marine soil that did not contain
lactic acid bacteria and left to stand, it was confirmed that the
lactic acid bacteria added therein were not destroyed and
proliferated.
[0110] Furthermore, the above-described lactic acid bacteria
(SIID17126-L1, SIID17126-L2, SIID21336-L1, SIID21336-L2 or
SIID21336-L3 alone or a mixture of at least two types of them) were
added to live seaweed that was farm-raised in the seawater. As a
result, the lactic acid bacteria were detected from the
seaweed.
[0111] In addition, as also confirmed in the fertilizer of (2)
above, the lactic acid bacteria were not destroyed even in the
soil, to which the above-described lactic acid bacteria
(SIID17126-L1, SIID17126-L2, SIID21336-L1, SIID21336-L2 or
SIID21336-L3 alone or a mixture of at least two types of them) were
added.
[0112] Therefore, it can be understood that the above-described
lactic acid bacteria (SIID17126-L1, SIID17126-L2, SIID21336-L1,
SIID21336-L2 or SIID21336-L3 alone or a mixture of at least two
types of them) can be administered with water, soil or the like. In
addition, the lactic acid bacteria (SIID17126-L1, SIID17126-L2,
SIID21336-L1, SIID21336-L2 or SIID21336-L3 alone or a mixture of at
least two types of them) can be used as a live bacterial
preparation that can be administered as one of environmental
measures to protect/maintain/restore/create biotope for human to
ecologically live with plants, animals, organisms, etc.
(4) Food and/or Drink
[0113] the above-described lactic acid bacteria (SIID17126-L1,
SIID17126-L2, SIID21336-L1, SIID21336-L2 or SIID21336-L3 alone or a
mixture of at least two types of them) was added to gastric acid
and left it to stand. As a result, it was confirmed that the added
lactic acid bacteria were not destroyed and remained live.
[0114] Therefore, it can be understood that the above-described
lactic acid bacteria (SIID17126-L1, SIID17126-L2, SIID21336-L1,
SIID21336-L2 or SIID21336-L3 alone or a mixture of at least two
types of them) may be added to food and/or drink. In addition, the
above-described lactic acid bacteria (SIID17126-L1,
[0115] SIID17126-L2, SIID21336-L1, SIID21336-L2 or SIID21336-L3
alone or a mixture of at least two types of them) can be similarly
added to medicines, dietary supplements, etc.
(5) Other Conditions
[0116] The above-described lactic acid bacteria (SIID17126-L1,
SIID17126-L2, SIID21336-L1, SIID21336-L2 or SIID21336-L3 alone or a
mixture of at least two types of them) may be applicable to a
method of producing fish products such as sashimi (thin slices of
fresh raw fish to eat as is) containing the lactic acid bacteria by
extracting them from fish. In addition, it is also achievable to
detoxify puffer toxin and it can be further applicable to a method
of processing fish containing unsaturated fatty acid(s), nitrogen,
phosphorus and/or potassium.
Furthermore, it can be also used in live bacterial preparations,
fertilizers, feed, food, etc. as lactic acid bacteria containing
five elements contained in fish (calcium, magnesium, nitrogen,
phosphorus and potassium) and trace elements (iron, chlorine,
manganese, zinc, copper, boron, molybdenum, and nickel).
* * * * *
References